Glutamine Metabolism Supports the Functional Activity of Immune Cells against Aspergillus fumigatus

Abstract

The reprogramming of cellular metabolism of immune cells is an essential process in the regulation of antifungal immune responses. In particular, glucose metabolism has been shown to be required for protective immunity against infection with Aspergillus fumigatus. However, given the intricate cross talk between multiple metabolic networks and signals, it is likely that cellular metabolic pathways other than glycolysis are also relevant during fungal infection. In this study, we demonstrate that glutamine metabolism is required for the activation of macrophage effector functions against A. fumigatus. Glutamine metabolism was found to be upregulated early after fungal infection and glutamine depletion or the pharmacological inhibition of enzymes involved in its metabolism impaired phagocytosis and the production of both proinflammatory and T-cell-derived cytokines. In an in vivo model, inhibition of glutaminase increased susceptibility to experimental aspergillosis, as revealed by the increased fungal burden and inflammatory pathology, and the defective cytokine production in the lungs. Moreover, genetic variants in glutamine metabolism genes were found to regulate cytokine production in response to A. fumigatus stimulation. Taken together, our results demonstrate that glutamine metabolism represents an important component of the immunometabolic response of macrophages against A. fumigatus both in vitro and in vivo. IMPORTANCE The fungal pathogen Aspergillus fumigatus can cause severe and life-threatening forms of infection in immunocompromised patients. The reprogramming of cellular metabolism is essential for innate immune cells to mount effective antifungal responses. In this study, we report the pivotal contribution of glutaminolysis to the host defense against A. fumigatus. Glutamine metabolism was essential both in vitro as well as in in vivo models of infection, and genetic variants in human glutamine metabolism genes regulated cytokine production in response to fungal stimulation. This work highlights the relevance of glutaminolysis to the pathogenesis of aspergillosis and supports a role for interindividual genetic variation influencing glutamine metabolism in susceptibility to infection.

Keywords: Aspergillus; antifungal immunity; glutamine; immunometabolism; macrophage.

FIG 1

A. fumigatus induces glutamine metabolism in macrophages. (A) Principal-component analysis of the glutamine gene signatures of macrophages left noninfected (Ctrl) or infected with A. fumigatus, with the proportion of variance per principal component indicated between parentheses. (B) Heatmap of the expression pattern of glutamine genes in macrophages left noninfected (Ctrl) or infected with A. fumigatus for 2 h (n = 3). Expression of glutamine genes is presented as centered, by hierarchical clustering, and with scaled log₂ fluorescence intensity (blue and red keys). (C) Glutamine consumption by macrophages left noninfected (Ctrl) or infected with A. fumigatus for 24 h (n = 3). (D) Levels of α-ketoglutarate, succinate, fumarate, malate, and glutamate (n = 3) in macrophages left noninfected (Ctrl) or infected with A. fumigatus for 6 h. Data are expressed as mean values ± SEM; *, P < 0.05, ***, P < 0.001 by two-way ANOVA with Tukey’s multiple-comparison test or Student’s two-tailed t test.

FIG 2

Glutamine depletion impairs antifungal effector functions of macrophages. (A) Levels of IL-1β, IL-6, and TNF secreted by macrophages left noninfected (Ctrl) or infected for 24 h with A. fumigatus in media without or with 2 mM or 5 mM glutamine (n = 4). (B) Levels of IFN-γ and IL-22 secreted by PBMCs left non-stimulated (Ctrl) or stimulated for 7 days with A. fumigatus in media without or with 2 mM or 5 mM glutamine (n = 4). (C) Lactate secretion by macrophages left noninfected (Ctrl) or infected for 24 h in media without or with 2 mM or 5 mM glutamine (n = 4). (D) Phagocytosis (n = 4) and (E) conidiacidal activity (n = 4) of macrophages in media without or with 2 mM or 5 mM glutamine. (E) Levels of cytosolic ROS (DHE, left) and mitochondrial ROS (Mitosox, right) by macrophages left noninfected (Ctrl) or infected for 4 h in media without or with 2 mM or 5 mM glutamine. Data are expressed as mean values ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student’s two-tailed t test.

FIG 3

Schematic diagram of the glutamine metabolism pathways and the site of action of inhibitors.

FIG 4

Pharmacological inhibition of glutamine metabolism at different steps impairs macrophage responses to A. fumigatus. (A) Levels of IL-1β, IL-6, and TNF secreted by macrophages infected for 24 h with A. fumigatus (n = 4), and (B) levels of IFN-γ and IL-22 secreted by PBMCs stimulated for 7 days with A. fumigatus (n = 4) and left untreated (-) or treated with GPNA, DON or BPTES. Uninfected cells were used as control (Ctrl). (C) Lactate secretion by macrophages infected for 24 h with A. fumigatus, and left untreated (-) or treated with GPNA, DON or BPTES (n = 4). Uninfected cells were used as control (Ctrl). (D) Phagocytosis and (E) conidiacidal activity of macrophages infected with A. fumigatus, left untreated (-) or treated with GPNA, DON or BPTES (n = 4). (F) Levels of cytosolic ROS (left) and mitochondrial ROS (right) in macrophages infected for 4 h with A. fumigatus and left untreated (-) or treated with GPNA, DON or BPTES (n = 4). Uninfected cells were used as control (Ctrl). Data are expressed as mean values ± SEM; *, P < 0.05; **, P < 0.01 by Student’s two-tailed t test.

FIG 5

Glutamine synthase inhibition in vivo increases susceptibility to aspergillosis. (A) mRNA expression of Gls, Got2, Gpt, Slc7a5, and Slc1a5 in the lungs of mice after 3 days of infection with A. fumigatus (n = 6). Naïve mice were used as control (Ctrl). (B) Fungal burden (log₁₀) per gram of lung tissue was determined after 3 days of infection in mice treated with vehicle (PBS), l-glutamine supplementation or BPTES (n = 6, representative of three independent experiments). (C) Representative H&E-stained lung sections of infected mice treated with vehicle (PBS), l-glutamine supplementation or BPTES. The graph indicates the area of inflammation (%) within the whole tissue section. (D) Levels of IL-1β, IL-6, TNF, IFN-γ, and IL-22 (n = 6) and (E) lactate (n = 6) in lung homogenates of infected mice treated with vehicle (PBS), l-glutamine supplementation or BPTES. Data are expressed as mean values ± SEM; *, P < 0.05; **, P < 0.01 by Student’s two-tailed t test.